title: Regioselective hydroxylation of C(12)-C(15) fatty acids with fluorinated substituents by cytochrome P450 BM3.
abstract: We demonstrate herein that wild-type cytochrome P450 BM3 can recognize non-natural substrates, such as fluorinated C12 -C15 chain-length fatty acids, and show better catalysis for their efficient conversion. Although the binding affinities for fluorinated substrates in the P450 BM3 pocket are marginally lower than those for non-fluorinated substrates, spin-shift measurements suggest that fluoro substituents at the ω-position can facilitate rearrangement of the dynamic structure of the bulk-water network within the hydrophobic pocket through a micro desolvation process to expel the water ligand of the heme iron that is present in the resting state. A lowering of the Michaelis-Menten constant (Km ), however, indicates that fluorinated fatty acids are indeed better substrates compared with their non-fluorinated counterparts. An enhancement of the turnover frequencies (kcat ) for electron transfer from NADPH to the heme iron and for CH bond oxidation by compound I (Cpd I) to yield the product suggests that the activation energies associated with going from the enzyme-substrate (ES state) to the corresponding transition state (ES(≠) state) are significantly lowered for both steps in the case of the fluorinated substrates. Delicate control of the regioselectivity by the fluorinated terminal methyl groups of the C12 -C15 fatty acids has been noted. Despite the fact that residues Arg47/Tyr51/Ser72 exert significant control over the hydroxylation of the subterminal carbon atoms toward the hydrocarbon tail, the fluorine substituent(s) at the ω-position affects the regioselective hydroxylation. For substrate hydroxylation, we have found that fluorinated lauric acids probably give a better structural fit for the heme pocket than fluorinated pentadecanoic acid, even though pentadecanoic acid is by far the best substrate among the reported fatty acids. Interestingly, 12-fluorododecanoic acid, with only one fluorine atom at the terminal methyl group, exhibits a comparable turnover frequency to that of pentadecanoic acid. Thus, fluorination of the terminal methyl group introduces additional interactions of the substrate within the hydrophobic pocket, which influence the electron transfers for both dioxygen activation and the controlled oxidation of aliphatics mediated by high-valent oxoferryl species.
authors.affiliation: Institute of Chemistry, Academia Sinica, Taipei 115 (Taiwan), Fax: (+886) 2-2783-1237; Department of Chemistry, National Cheng Kung University, Tainan 701 (Taiwan).
authors.forename: Chih-Hsiang
authors.initials: CH
authors.lastname: Chiang
authors.forename: Ravirala
authors.initials: R
authors.lastname: Ramu
authors.forename: Yi-Jung
authors.initials: YJ
authors.lastname: Tu
authors.forename: Chung-Ling
authors.initials: CL
authors.lastname: Yang
authors.forename: Kok Yaoh
authors.initials: KY
authors.lastname: Ng
authors.forename: Wen-I
authors.initials: WI
authors.lastname: Luo
authors.forename: Charles H
authors.initials: CH
authors.lastname: Chen
authors.forename: Yu-Ying
authors.initials: YY
authors.lastname: Lu
authors.forename: Chen-Lun
authors.initials: CL
authors.lastname: Liu
authors.forename: Steve S-F
authors.initials: SS
authors.lastname: Yu
mesh_terms: D003577:Cytochrome P-450 Enzyme System; D005227:Fatty Acids / Q000737:chemistry*; D005466:Fluorocarbons; D054879:Halogenation; D006860:Hydrogen Bonding; D006900:Hydroxylation; D007850:Lauric Acids / Q000737:chemistry*; D010084:Oxidation-Reduction
chemical_list: D005227:Fatty Acids; D005466:Fluorocarbons; D007850:Lauric Acids; D003577:Cytochrome P-450 Enzyme System; C117025:pentadecanoic acid; C522391:perfluorododecanoic acid
keywords: CH activation; cytochrome P450; enzyme catalysis; fluorinated substituents; van der Waals interactions